The EBV membrane antigen (EBV-MA) gp350 contains virus neutralizing epitopes and is currently being tested as a possible EBV vaccine candidate in England. A series of 15 monoclonal antibodies (mAbs) to EBV-MA were obtained from Louis Qualtiere and Gary Pearson. These have been used to screen antigen expressing clones for reactivity to different epitopes in order to identify the nucleic acid sequences important for virus neutralization. Initially ten overlapping clones which express different portions of unglycosylated gp350 were constructed in E. coli. Four of fifteen mAbs tested reacted with the recombinant E. coli antigens, and three antigenic epitopes (nucleotides 1980-2307, 3186-3528 and 3528-3576 in Bam HI L) identified. Proteins expressed by the clones were used to produce antisera in rabbits. When tested for neutralization of EBV in tissue culture, the rabbit antisera as well as the mAbs which recognized the three epitopes failed to neutralize, suggesting that these epitopes are not involved in neutralization. A manuscript describing these data has been published in J. Gen. Virol. Since the remaining mAbs may only recognize the glycosylated form of gp350, the entire gp350 coding sequence has also been expressed in a Baculovirus expression system which produces glycosylated protein. All of the MAbs react with the baculovirus expressed protein. Immunofluorescence studies of Sf9 cells expressing gp350 suggest the protein is membrane associated. Five additional subclones which express different portions of gp350 in the Baculovirus expression system have been prepared and used for additional epitope mapping studies. Recent data indicate that these subgenomic proteins do not react with all the mAbs recognized by the entire Baculovirus- expressed gp350.